Oligosaccharide dehydrogenase-catalyzed assay for the determination of polysaccharides

Oligosaccharide dehydrogenase (ODH), an enzyme known to have a broad select ivity for reducing sugars of low molecular weight, was investigated to dete rmine its catalytic properties with larger polysaccharides. Six substrates were studied: pullulan standards with molecular weights of between 5,400 an d 90,900, debranched starch, and dextran. In addition, maltotriose, isomalt otriose, maltose, and glucose were used as substrates for comparison. ODH c atalyzed the oxidation of the large pullulans with a degree of polymerizati on of at least 560. Isomaltotriose and dextran were not oxidized. ODH activ ity for the pullulans, expressed as the rate constant K-ps, was only three times lower than that for maltose. When the oxidation of sugars with ODH wa s coupled to a color-forming reaction, quantitative spectrophotometric dete rmination of sugars was possible using either Meldola's blue or N-methylphe nazinium as electron accepters in combination with nitrotetrazolium blue. L inear calibration curves for maltose, maltotriose, and debranched starch we re obtained using this ODH method and compared with curves from the convent ional spectrophotometric copper sulfate method. This work demonstrates that ODH can be advantageously used for the determination of polysaccharides. ( C) 1998 Academic Press.